Hepatocyte delivery vehicle for delivery of glargine insulin to a mammal

ABSTRACT

The instant invention is drawn to a hepatocyte targeted composition comprising a mixture of free glargine insulin and glargine insulin associated with a water insoluble target molecule complex, wherein the complex comprises multiple linked individual units and a supra-molecular lipid construct matrix. Glargine insulin is present within the complex in at least one form wherein the glargine insulin has a positive charge which interacts with a negative charge on the complex. The invention also includes methods for the manufacture of the composition and methods of managing blood glucose levels in individuals with Type I and Type II diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/313,828, filed May 18, 1999, which claims priority to U.S.Provisional Patent Application No. 60/085,969, filed May 19, 1998, eachof which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Diabetes is a disorder affecting large numbers of people worldwide.Management approaches to control Type I and Type II diabetes aimprimarily at normalizing blood glucose levels to prevent short- andlong-term complications. Many patients require multiple daily injectionsof an insulin to control their diabetes. Several insulin products havebeen produced that control blood sugar levels over differing timeintervals. Several products combine various forms of insulin in anattempt to provide a preparation which controls glucose levels over awider period of time.

Previous attempts to normalize blood glucose levels in Type I and TypeII diabetic patients have centered on the subcutaneous administration ofinsulin in various time-released formulations, such as Itralente andhumulin NPH insulin pharmaceutical products. These formulations haveattempted to delay and subsequently control the bio-distribution ofinsulin by regulating release of insulin to peripheral tissues with theexpectation that sustained management of insulin bio-availability willlead to better glucose control. Glargine insulin is a long-acting formof insulin in which insulin is released from the subcutaneous tissuearound the site of injection into the bloodstream at a relativelyconstant rate throughout the day. Although glargine insulin is releasedat a constant rate throughout the day, the released insulin reaches awide range of systems within the body rather than being delivered totargeted areas of the body. What is needed is a composition of insulinwhere a portion of the dosed insulin is released at a relativelyconstant rate throughout the day and another portion of insulin that istime released from the site of administration and targeted for deliveryto the liver to better control glucose production.

There is, therefore, an unmet need in the art for compositions andmethods of managing blood glucose levels in Type I and Type II diabeticpatients. The present invention meets these needs by providing along-acting composition comprising glargine insulin that is free andglargine insulin that is associated with a supra-molecular lipidconstruct targeted for delivery to hepatocytes. A supra-molecular lipidconstruct is a lipid/phospholipid particle in which individual lipidmolecules cooperatively interact to create a bipolar lipid membranewhich encloses and isolates a portion of the medium in which it wasformed. The supra-molecular lipid construct releases free glargine overtime as well as targets a portion of the remaining glargine insulin tothe hepatocytes in the liver to better control glucose storage andproduction.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention includes a hepatocyte-targetingcomposition comprising: free glargine insulin and glargine insulinassociated with a water-insoluble target molecule complex; wherein thetarget molecule complex is comprised of a combination of: multiplelinked individual units, the individual units comprising: at least onebridging component selected from the group consisting of a transitionelement, an inner transition element, and a neighbor element of thetransition element; and a complexing component; and a supra-molecularlipid construct matrix comprising at least one lipid component; providedthat when the transition element is chromium, a chromium target moleculecomplex is created; wherein the target molecule complex comprises anegative charge.

In another aspect, the composition further comprises at least one freenon-glargine insulin and at least one non-glargine insulin associatedwith a water-insoluble target molecule complex.

In yet another aspect, non-glargine insulin is selected from the groupconsisting of lispro insulin, aspart insulin, regular insulin, lenteinsulin, ultralente insulin, recombinant human regular insulin,recombinant human insulin isophane or premixed combinations of any ofthe aforementioned insulins, and a combination of two or more of theaforementioned insulins.

In yet another aspect, non-glargine insulin comprises insulin-likemoieties, including fragments of insulin molecules, that have thebiological activity of insulins.

In still another aspect, the lipid component comprises at least onelipid selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dimyristoyl-sn-glycero-3-phosphate.

In yet another aspect, the lipid component comprises at least one lipidselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dicetylphosphate.

In one aspect, the lipid component comprises a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and dicetylphosphate.

In another aspect, the bridging component is chromium.

In still another aspect, the complexing component comprises at least onemember selected from the group consisting of:

-   N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   aminopyrrol iminodiacetic acid;-   N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic acid;-   benzimidazole methyl iminodiacetic acid;-   N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid;-   N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiacetic    acid; and-   N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid.

In yet another aspect, the complexing component comprisespoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid].

In one aspect, the present invention includes a method of manufacturinga hepatocyte targeted composition of the invention comprising: creatinga target molecule complex, wherein the complex comprises multiple linkedindividual units and a supra-molecular lipid construct matrix; forming asuspension of the target molecule complex in water; adjusting the pH ofthe water suspension to approximately pH 5.3; adjusting the pH ofglargine insulin to approximately pH 4.8; and combining glargine insulinand the target molecule complex.

In another aspect, a method of treating a patient for Type I or Type IIdiabetes comprises administering to the patient an effective amount of ahepatocyte targeted composition of the invention.

In still another aspect, the route of administration is selected fromthe group consisting of oral, parenteral, subcutaneous, pulmonary andbuccal.

In yet another aspect, the route of administration is oral orsubcutaneous.

In another aspect, the non-glargine insulin is selected from the groupconsisting of lispro insulin, aspart insulin, regular insulin, lenteinsulin, ultralente insulin, recombinant human regular insulin,recombinant human insulin isophane or premixed combinations of any ofthe aforementioned insulins, and a combination of two or more of theaforementioned insulins.

In still another aspect, a method of increasing the bioavailability ofglargine insulin in a patient comprises: administering glargine insulinin a hepatocyte-targeting composition, the composition comprising freeglargine insulin and glargine insulin associated with a water insolubletarget molecule complex, wherein the complex comprises multiple linkedindividual units and a supra-molecular lipid construct matrix containinga negative charge, the multiple linked individual units comprising: (a)a bridging component selected from the group consisting of a transitionelement, an inner transition element, a neighbor element of thetransition element and a mixture of any of the foregoing elements, (b) acomplexing component, provided that when the transition element ischromium, a chromium target molecule complex is created, wherein themultiple linked individual units are combined with the supra-molecularlipid construct matrix, wherein the insulins are associated with thetarget molecule complex that contains a negative charge; thereby theassociation between glargine insulin and the water insoluble targetmolecule complex is altered within the patient to form new structuresassociated with the glargine insulin, wherein the new structures arepresent in soluble and insoluble forms and are delivered to sites ofinsulin activity.

In one aspect, the present invention comprises delivery of glargineinsulin to fat, liver, and muscle.

In another aspect, delivery of glargine insulin to sites of insulinactivity occurs over a plurality of meals.

In still another aspect, delivery of a hepatic component occurs atmeal-time.

In yet another aspect, a kit for treating Type I or Type II diabetes ina mammal is provided, the kit comprising glargine insulin and a waterinsoluble target molecule complex, wherein the complex comprisesmultiple linked individual units and a supra-molecular lipid constructmatrix containing a negative charge, the multiple linked individualunits comprising: a bridging component selected from the groupconsisting of a transition element, an inner transition element, aneighbor element of the transition element and a mixture of any of theforegoing elements, and a complexing component, provided that when thetransition element is chromium, a chromium target molecule complex iscreated, wherein the multiple linked individual units are combined withthe supra-molecular lipid construct matrix, wherein the glargine insulinis associated with a target molecule complex, wherein said complexcomprises a negative charge, the kit further comprising a physiologicalbuffered solution, an applicator, and an instructional material for theuse thereof.

In one aspect, a kit further comprises at least one non-glargineinsulin.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, there is depicted in thedrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 is a depiction of the chemical structure of glargine insulin.

FIG. 2 is a depiction of a pharmaceutical composition that combines freeglargine insulin and glargine insulin associated with a water insolubletarget molecule complex.

FIG. 3 is an outline of the method of manufacturing a hepatocytetargeted pharmaceutical composition that combines free glargine insulinand glargine insulin associated with a water insoluble target moleculecomplex.

FIG. 4 is a graph of the concentrations of glucose in blood ofindividual patients treated once before breakfast with HDV-glargineinsulin.

FIG. 5 is a graph of the effect of a single dose of HDV-glargine insulinon average blood glucose concentrations in patients consuming threemeals during the day.

FIG. 6 is a graph of the effect of HDV-glargine insulin on blood glucoseconcentrations over time relative to blood glucose concentrations duringfasting.

DETAILED DESCRIPTION OF THE INVENTION

This invention includes a hepatocyte targeted pharmaceutical compositionthat combines free glargine insulin and glargine insulin associated witha water insoluble target molecule complex targeted to hepatocytes in theliver of a patient to provide an effective means of managing bloodglucose levels. The composition can be administered subcutaneously ororally for the purpose of normalizing blood glucose levels in patientsaffected with abnormal glucose utilization.

The invention further provides a method of manufacturing a compositioncomprising free glargine insulin and glargine insulin associated with awater insoluble target molecule complex that targets delivery of thecomplex to the hepatocytes. The target molecule complex comprises asupra-molecular lipid construct matrix containing multiple linkedindividual units of a structure formed by a metal complex.

Additionally, the invention provides methods of managing blood glucoselevels in individuals with Type I and Type II diabetes by administeringan effective dose of a hepatocyte targeted pharmaceutical compositionthat combines free glargine insulin and glargine insulin associated witha water insoluble target molecule complex targeted for delivery tohepatocytes. The combination of free glargine insulin and glargineinsulin associated with a water insoluble target molecule complexcreates a dynamic equilibrium process between the two forms of insulinthat occurs in vivo to help control the movement of free glargineinsulin to the receptor sites of hormonal action, such as the muscle andadipose tissue of a diabetic patient over a designated time period.Hepatocyte targeted glargine insulin is also delivered to the liver of adiabetic patient over a different designated time period than freeglargine insulin thereby introducing new pharmacodynamic profiles ofinsulin when free glargine insulin is released from the supra-molecularlipid construct. In addition, a portion of glargine insulin that isassociated with the supra-molecular construct is targeted to the liver.This new pharmacodynamic profile of the product provides not onlylong-acting basal insulin for peripheral tissues, but also meal-timehepatic insulin stimulation for the management of hepatic glucosestorage during a meal. Free glargine insulin is released from the siteof administration and is distributed throughout the body. Glargineinsulin associated with a water insoluble target molecule complex isdelivered to the liver, where it is released over time from the complex.The rate of release of glargine insulin associated with the targetmolecule complex is different than the rate of release of free glargineinsulin from the site of administration. These different release ratesof insulin delivery, combined with the targeted delivery of insulinassociated with a supra-molecular lipid construct to the liver, providefor the normalization of glucose concentrations in patients with Type Iand Type II diabetes. The hepatocyte targeted composition can alsocomprise other types of insulin, or a combination of other types ofinsulin.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in organicchemistry and protein chemistry are those well known and commonlyemployed in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “active ingredient” refers to glargine insulin and otherinsulins.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, as indicated in thefollowing table: Full Name Three-Letter Code Alanine Ala Arginine ArgAsparagine Asn Aspartic Acid Asp Cysteine Cys Cystine Cys—Cys GlutamicAcid Glu Glutamine Gln Glycine Gly Histidine His Isoleucine Ile LeucineLeu Lysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine SerThreonine Thr Tryptophan Trp Tyrosine Tyr Valine Val

The term “lower” means the group it is describing contains from 1 to 6carbon atoms.

The term “alkyl”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon having the number of carbon atoms designated (i.e. C₁-C₆means one to six carbons) and includes straight, branched chain orcyclic groups. Examples include: methyl, ethyl, propyl, isopropyl,butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl andcyclopropylmethyl. Most preferred is (C₁-C₃) alkyl, particularly ethyl,methyl and isopropyl.

The term “alkylene”, by itself or as part of another substituent means,unless otherwise stated, a straight, branched or cyclic chainhydrocarbon having two substitution sites, e. g., methylene (—CH₂—),ethylene (—CH₂CH₂—), isopropylene (—CH(CH₃)═CH₂), etc.

The term “aryl”, employed alone or in combination with other terms,means, unless otherwise stated, a cyclic carbon ring structure, with orwithout saturation, containing one or more rings (typically one, two orthree rings) wherein such rings may be attached together in a pendantmanner, such as a biphenyl, or may be fused, such as naphthalene.Examples include phenyl; anthracyl; and naphthyl. The structure can haveone or more substitution sites where functional groups, such as alcohol,alkoxy, amides, amino, cyanides, halogen, and nitro, are bound.

The term “arylloweralkyl” means a functional group wherein an aryl groupis attached to a lower alkylene group, e.g., —CH₂CH₂-phenyl.

The term “alkoxy” employed alone or in combination with other termsmeans, unless otherwise stated, an alkyl group or an alkyl groupcontaining a substituent, such as a hydroxyl group, having thedesignated number of carbon atoms connected to the rest of the moleculevia an oxygen atom, such as, for example, —OCHOH—, —OCH₂OH, methoxy(—OCH₃), ethoxy (—OCH₂CH₃), 1-propoxy (—OCH₂CH₂CH₃), 2-propoxy(isopropoxy), butoxy (—OCH₂CH₂CH₂CH₃), pentoxy (—OCH₂CH₂CH₂CH₂CH₃), andthe higher homologs and isomers.

The term “acyl” means a functional group of the general formula—C(═O)—R, wherein —R is hydrogen, hydrocarbyl, amino or alkoxy. Examplesinclude acetyl (—C(═O)CH₃), propionyl (—C(═O)CH₂CH₃), benzoyl(—C(═O)C₆H₅), phenylacetyl (—C(═O)CH₂C₆H₅), carboethoxy (—CO₂ CH₂CH₃),and dimethylcarbamoyl (—C(═O)N(CH₃)₂).

The terms “halo” or “halogen” by themselves or as part of anothersubstituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom.

The term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself oras part of another substituent means, unless otherwise stated, anunsubstituted or substituted, stable, mono- or multicyclic heterocyclicring system which consists of carbon atoms and at least one heteroatomselected from the group consisting of N, O, and S, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen atom may be optionally quaternized. The heterocyclic system maybe attached, unless otherwise stated, at any heteroatom or carbon atomwhich affords a stable structure. Examples include pyrrole, imidazole,benzimidazole, phthalein, pyridenyl, pyranyl, furanyl, thiazole,thiophene, oxazole, pyrazole, 3-pyrroline, pyrrolidene, pyrimidine,purine, quinoline, isoquinoline, carbazole, etc.

The term “chromium target molecule complex” refers to a complexcomprising a plurality of chromium (Cr) atoms capable of accepting up tosix ligands contributed by multivalent molecules, such as ligands fromnumerous molecules of N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid forming a complicated polymeric structure linked in athree-dimensional array. The complex is linked in a polymeric structurethat is insoluble in water but soluble in organic solvents.

The term “supra-molecular lipid construct” refers to a lipid and/orphospholipid particle in which individual lipid molecules cooperativelyinteract to create a bipolar lipid membrane that encloses and isolates aportion of the medium in which the construct resides.

A “complexing agent” is a compound that forms a complex with a selectedmetal bridging agent, e. g. a salt of chromium, zirconium, etc., thatexhibits polymeric properties. The polymeric complex is substantiallyinsoluble in water and soluble in organic solvents.

By “substantially insoluble” is meant that a polymeric complex, such asa polymeric chromium target molecule complex or other metal targetingcomplexes, exhibits the property of being insoluble in water at roomtemperature. Such a polymeric complex, which may be crystalline,amorphous in composition, or a dissociated form thereof, when associatedwith a supra-molecular lipid construct forms a transport agent thatcarries and delivers glargine insulin to hepatocytes in the liver.

The term “associated with” means that the referenced material isincorporated into or on the surface of, or within the supra-molecularlipid construct matrix.

The terms “glargine” and “glargine insulin” both refer to a recombinanthuman insulin analog which differs from human insulin in that the aminoacid asparagine at position A21 is replaced by glycine and two argininesare added to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063. Thestructural formula of glargine insulin is provided in FIG. 1.

The term “non-glargine insulin” refers at all insulins, either naturalor recombinant that are not glargine insulin. The term includesinsulin-like moieties, including fragments of insulin molecules, thathave biological activity of insulins. Examples of non-glargine insulinsinclude, but are not limited to recombinant human regular insulin,recombinant human insulin isophane, recombinant human regular insulin,insulin aspart, insulin lispro, insulin lente, and insulin ultralente.

The term “free insulin” refers to an insulin that is not associated witha target molecule complex.

“HDV”, or “Hepatocyte Delivery Vehicle”, is a water insoluble targetmolecule complex comprising a supra-molecular lipid construct matrixcontaining multiple linked individual units of a structure formed by thecombination of a metal bridging agent and a complexing agent. “HDV” isdescribed in WO 99/59545, Targeted Liposomal Drug Delivery System.

“HDV-glargine” is a designation for a hepatocyte targeted compositioncomprising a mixture of free glargine insulin and glargine insulinassociated with a water insoluble target molecule complex, wherein thecomplex comprises multiple linked individual units of chromium andN-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, formed bythe combination of a metal bridging agent and a complexing agent, and asupra-molecular lipid construct matrix.

The term “treat” means to reduce the frequency with which symptoms of adisease, disorder, or adverse condition, and the like, are experiencedby a patient.

The term “pharmaceutically acceptable carrier” means a chemicalcomposition with which the active ingredient may be combined and which,following the combination, can be used to administer the activeingredient to a subject.

The term “physiologically acceptable” means that the ingredient is notdeleterious to the subject to which the composition is to beadministered.

Description of the Invention—Composition

The structure of glargine insulin is provided in FIG. 1. Glargineinsulin differs from human insulin in that glargine insulin has amolecular structure that replaces asparagine with glycine at theC-terminal end of the A chain of human insulin and adds the dipeptide ofarginine at the C-terminal end of the B chain of human insulin. Theisoelectric point of a compound is the pH at which the overall charge ofthe compound is neutral. However, regions of negative and positivecharges still remain within the compound. The isoelectric point of humaninsulin is at pH 5.3. The isoelectric point of glargine insulin ishigher than human insulin because the amino acid substitutions inglargine insulin raise the isoelectric point of glargine insulin to pH5.8-6.2. Compounds are generally less soluble in aqueous solutions at pHranges around the isoelectric point. A compound is generally moresoluble in aqueous systems where the pH of the solution is approximately1-2 pH units higher or lower than the isoelectric point. The higherisoelectric point allows glargine insulin to remain soluble in a mildlyacidic environment over a broader pH range.

LANTUS® (insulin glargine [rDNA origin] injection) is a sterile solutionof glargine insulin for use as an injectable insulin for diabeticpatients for subsequent management of glucose levels in vivo. Glargineinsulin is a recombinant human insulin analog that is a long-acting (upto 24-hour duration of action), parenteral blood-glucose-lowering agent.LANTUS is produced by recombinant DNA technology utilizing anon-pathogenic laboratory strain of Escherichia coli (K12) as theproduction organism. LANTUS consists of glargine insulin dissolved in aclear aqueous fluid. Each milliliter of LANTUS (insulin glargineinjection) contains 100 IU (3.6378 mg) glargine insulin, 30 mcg zinc,2.7 mg m-cresol, 20 mg glycerol 85%, and water for injection. The pH ofcommercially available LANTUS insulin can be adjusted by addition ofaqueous solutions of acids, bases or buffers that are physiologicallycompatible. LANTUS has a pH of approximately 4.

A depiction of a pharmaceutical composition that combines free glargineinsulin and glargine insulin associated with a target molecule complexis shown in FIG. 2. The target molecule complex comprises multiplelinked individual units formed by complexing a bridging component with acomplexing agent. The bridging component is a water soluble salt of ametal capable of forming a water-insoluble coordinated complex with acomplexing agent. A suitable metal is selected from the transition andinner transition metals or neighbors of the transition metals. Thetransition and inner transition metals from which the metal can beselected are: Sc (scandium), Y (yttrium), La (lanthanum), Ac (actinium),the actinide series; Ti (titanium), Zr (zirconium), Hf (hafnium), V(vanadium), Nb (niobium), Ta (tantalum), Cr (chromium), Mo (molybdenum),W (tungsten), Mn (manganese), Tc (technetium), Re (rhenium), Fe (iron),Co (cobalt), Ni (nickel), Ru (ruthenium), Rh (rhodium), Pd (palladium),Os (osmium), Ir (iridium), and Pt (platinum). The neighbors of thetransition metals from which the metal can be selected are: Cu (copper),Ag (silver), Au (gold), Zn (zinc), Cd (cadmium), Hg (mercury), Al(aluminum), Ga (gallium), In (indium), Tl (thallium), Ge (germanium), Sn(tin), Pb (lead), Sb (antimony) and Bi (bismuth), and Po (polonium).Examples of metal compounds useful as bridging agents include chromiumchloride (III) hexahydrate; chromium (III) fluoride tetrahydrate;chromium (III) bromide hexahydrate; zirconium (IV) citrate ammoniumcomplex; zirconium (IV) chloride; zirconium (IV) fluoride hydrate;zirconium (IV) iodide; molybdenum (III) bromide; molybdenum (III)chloride; molybdenum (IV) sulfide; iron (III) hydrate; iron (III)phosphate tetrahydrate and iron (III) sulfate pentahydrate.

The complexing agent is a compound that forms a water insolublecoordinated complex with a bridging component. There are severalfamilies of suitable complexing agents.

A complexing agent can be selected from the family of iminodiaceticacids of the formula (1) where R₁ is loweralkyl, aryl, arylloweralkyl,and a heterocyclic substituent.

Suitable compounds of the formula (1) include:

-   N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;-   N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;-   aminopyrrol iminodiacetic acid;-   N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic acid;-   benzimidazole methyl iminodiacetic acid;-   N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid;-   N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiacetic    acid; and-   N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid and    other derivatives of N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl)    iminodiacetic acid of formula (2),

where R₂ and R₃ are the following: R₂ R₃ H iso-C₄H₉ H CH₂CH₂SCH₃ HCH₂C₆H₄-p-OH CH₃ CH₃ CH₃ iso-C₄H₉ CH₃ CH₂CH₂SCH₃ CH₃ C₆H₅ CH₃ CH₂C₆H₅CH₃ CH₂C₆H₄-p-OCH₃

A complexing agent can be selected from the family of imino diacidderivatives of the general formula (3), where R₄, R₅, and R₆ areindependent of each other and can be hydrogen, loweralkyl, aryl,arylloweralkyl, alkoxyloweralkyl, and heterocyclic.

Suitable compounds of the formula (3) include: N′-(2-acetylnaphthyl)iminodiacetic acid (NAIDA); N′-(2-naphthylmethyl) iminodiacetic acid(NMIDA); iminodicarboxymethyl-2-naphthylketone phthalein complexone; 3β:7α: 12α: trihydroxy-24-norchol anyl-23-iminodiacetic acid; benzimidazolemethyl iminodiacetic acid; and N-(5,pregnene-3-β-ol-2-oylcarbamoylmethyl) iminodiacetic acid.

A complexing agent can be selected from the family of amino acids offormula (4),

where R₇ is an amino acid side chain, R₈ is loweralkyl, aryl,arylloweralkyl, and R₉ is pyridoxylidene.

Some suitable amino acids of the formula (4) are aliphatic amino acids,including glycine, alanine, valine, leucine, and isoleucine;hydroxyamino acids, including serine, and threonine; dicarboxylic aminoacids and their amides, including aspartic acid, asparagine, glutamicacid, and glutamine; amino acids having basic functions, includinglysine, hydroxylysine, histidine, and arginine; aromatic amino acids,including phenylalanine, tyrosine, tryptophan, and thyroxine; andsulfur-containing amino acids, including cystine and methionine. Otheramino acids and derivatives of biological importance include, but arenot necessarily limited to (3-alanine,y-amino) butyric acid,O-diazoacetylserine (azaserine), homoserine, omithine, citrulline, andpenicillamine.

Members of the pyridoxylidene class of complexing agents include, butare not limited to: pyridoxylidene glutamate; pyridoxylidene isoleucine;pyridoxylidene phenylalanine; pyridoxylidene tryptophan;pyridoxylidene-5-methyl tryptophan; pyridoxylidene-5-hydroxytryptamine;and pyridoxylidene-5-butyltryptamine.

A complexing agent can be selected from the family of diamines of thegeneral formula (6),

where R₁₀ is hydrogen, loweralkyl, or aryl; R₁₁ is loweralkylene orarylloweralky; R₁₂ and R₁₃ independently are hydrogen, loweralkyl,alkyl, aryl, arylloweralkyl, acylheterocyclic, toluene, sulfonyl ortosylate.

Some suitable complexing agent diamines of the formula (6) include, butare not limited to, ethylenediamine-N,N diacetic acid;ethylenediamine-N,N-bis (-2-hydroxy-5-bromophenyl) acetate;N′-acetylethylenediamine-N,N diacetic acid; N′-benzoylethylenediamine-N,N diacetic acid; N′-(p-toluenesulfonyl)ethylenediamine-N,N diacetic acid; N′-(p-t-butylbenzoyl)ethylenediamine-N,N diacetic acid; N′-(benzenesulfonyl)ethylenediamnine-N,N diacetic acid; N′-(p-chlorobenzenesulfonyl)ethylenediamine-N,N diacetic acid; N′-(p-ethylbenzenesulfonylethylenediamine-N,N diacetic acid; N′-acyl and N′-sulfonylethylenediamine-N,N-diacetic acid; N′-(p-n-propylbenzenesulfonyl)ethylenediamine-N,N diacetic acid; N′-(naphthalene-2-sulfonyl)ethylenediamine-N,N diacetic acid; and N′-(2,5-dimethylbenzenesulfonyl)ethylenediamine-N,N diacetic acid.

Other suitable complexing agents include: penicillamine;p-mercaptoisobutyric acid; dihydrothioctic acid; 6-mercaptopurine;kethoxal-bis(thiosemicarbazone); Hepatobiliary Amine Complexes,1-hydrazinophthalazine (hydralazine); sulfonyl urea; Hepatobiliary AminoAcid Schiff Base Complexes; pyridoxylidene glutamate; pyridoxylideneisoleucine; pyridoxylidene phenylalanine; pyridoxylidene tryptophan;pyridoxylidene 5-methyl tryptophan; pyridoxylidene-5-hydroxytryptamine;pyridoxylidene-5-butyltryptamine; tetracycline;7-carboxy-p-hydroxyquinoline; phenolphthalein; eosin I bluish; eosin Iyellowish; verograffin; 3-hydroxyl-4-formyl-pyridene glutamic acid; andAzo substituted iminodiacetic acid.

Suitable complexing agents include: hepatobiliary dye complexes, such asrose bengal; congo red; bromosulfophthalein; bromophenol blue; toluidineblue; and indocyanine green; hepatobiliary contrast agents, such asiodipamide; and ioglycamic acid; bile salts, such as bilirubin;cholgycyliodohistamine; and thyroxine; hepatobiliary thio complexes,such as penicillamine; p-mercaptoisobutyric acid; dihydrothiocytic acid;6-mercaptopurine; and kethoxal-bis (thiosemicarbazone); hepatobiliaryamine complexes, such as 1-hydrazinophthalazine (hydralazine); andsulfonyl urea; hepatobiliary amino acid Schiff Base complexes, includingpyridoxylidene-5-hydroxytryptamine; andpyridoxylidene-5-butyltryptamine; hepatobiliary protein complexes, suchas protamine; ferritin; and asialo-orosomucoid; and asialo complexes,such as lactosaminated albumin; immunoglobulins, G, IgG; and hemoglobin.

The three-dimensional structure made from combining bridging agents andcomplexing agents is described in WO 99/59545, which is incorporated byreference. In an embodiment, the bridging agent is a metal salt, such aschromium chloride hexahydrate, that forms a coordinated complex withcomplexing agents, such as N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid. The bridging agent and the complexing agents arecombined to form a complex comprising multiple linked units in athree-dimensional array. In a preferred embodiment, the complexcomprises multiple units of chromiumpoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid]linked together in a polymeric type structure. In an embodiment, thechromium target molecule complex substance is soluble in a mixture oflipids containing 1,2-distearoyl-sn-glycero-3-phosphocholine, dicetylphosphate and cholesterol.

The complex is incorporated within a supra-molecular lipid construct,comprised of lipids or groups of lipids, to form a water insolubletarget molecule complex, as described in WO 99/59545. A suitable lipid,or a mixture of lipids where lipid molecules function individually or incombination thereof, will dissolve the metal complex and form asupra-molecular lipid construct that incorporates the metal complextherein. A suitable lipid is selected from a group of lipids commonlyemployed to form supra-molecular lipid constructs. Suitable lipidsinclude 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphate, and a mixture of any of theforegoing lipids or appropriate derivative of these lipids. In apreferred embodiment the lipids are a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dicetylphosphate. The selected lipid, or mixture of lipids, is maintained insuspension in aqueous media to form a structure able to incorporateglargine insulin into the structure. In an embodiment, a mixture of thetarget molecule complex and the supra-molecular lipid construct is alsoprovided with a masking agent in intimate association therewith toprotect it from immunoreactive attack, such as by macrophages.

Adjustment of the pH of an aqueous solution surrounding thesupra-molecular lipid construct containing the target molecule complex,by the addition of acids, bases or buffers, results in a negative chargein the supra-molecular lipid construct structure. The pH range at whichthis occurs depends upon the composition of the lipids. A preferredlipid system is a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine,cholesterol and dicetylphosphate. This mixture forms a negativelycharged supra-molecular lipid construct structure under physiologicalconditions. The supra-molecular lipid construct exhibits hepatocytetargeting specificity, i.e. is specific for cellular hepatocytes,thereby allowing the construct to be targeted to the liver.

A pharmaceutical composition comprises a mixture of free glargineinsulin and glargine insulin associated with a water insoluble targetmolecule complex. Free glargine insulin is the material depicted inFIG. 1. Free glargine insulin is not associated with the target moleculecomplex and is soluble in water. The other form of glargine insulin inthe composition is associated with a water insoluble target moleculecomplex.

It has been discovered in the present invention that when theappropriate lipid components are formulated into a water insolubletarget molecule complex using Sterile Water for Injection, USP (SWI)that has been terminally pH adjusted to pH 3.95±0.2, the overallelectronic charge on the target molecule complex is predominatelynegative. Glargine insulin has a net positive charge at pH 5.2±0.5,which is below the isoelectric point of the protein. The positive chargeon glargine insulin at pH 5.2±0.5 allows for interaction of thepositively charged portion of glargine insulin with the negativelycharge portion of the target molecule complex. This results inpositively charged glargine insulin being attracted to the negativelycharged target molecule complex. Portions of the charged glargineinsulin become associated with charges on the lipids and the chargedglargine insulin moves within the lipids, while other charged glargineinsulin molecules are sequestered within the core volume of thesupra-molecular lipid construct after partitioning through the variouslipid moieties of the supra-molecular construct.

There is an equilibrium between free glargine insulin in solution andglargine insulin associated with the water insoluble target moleculecomplex. Because the interactions between glargine insulin and thetarget molecule complex involve equilibria, over time free glargineinsulin is able to further bind and partition into the lipid domainsand/or the central core volume of the water insoluble target moleculecomplex. In an embodiment, free glargine insulin can be transformed intotransitory lipid derivatives by adsorbing onto, or reacting with,individual molecules of lipid that are in equilibrium with the waterinsoluble target molecule complex. These derivatives associate with thelipids of the water insoluble target molecule complex and enter thecore-volume of the complex, thus affecting the pharmacological activityof the product.

When a composition of the present invention is administered byinjection, the pharmacological activity of the composition in terms ofbioavailability will be realized when the supra-molecular lipidconstruct is located in the subcutaneous depot in vivo at pH 5.8-pH 6.2,where free glargine insulin is precipitated in an insoluble form. Therelease of free insulin from the supra-molecular lipid construct iscontrolled by a biokinetic release mechanism. The targetedsupra-molecular lipid construct with the remaining insulin is alsocontrolled by a biokinetic release mechanism regulated by an equilibriumbetween the insoluble to soluble forms of glargine insulin in thesubcutaneous depot as insoluble insulin solubilizes in response tophysiological conditions.

Description of the Invention—Method of Manufacture

FIG. 3 demonstrates an outline for a process for manufacturing a mixtureof free glargine insulin and glargine insulin associated with a waterinsoluble target molecule complex.

In an embodiment, the manufacture of the composition involves threeoverall steps: preparing a target molecule complex, incorporating thetarget molecule complex into a supra-molecular lipid construct, andcombining the target molecule complex with glargine insulin to form apharmaceutical composition.

The target molecule complex comprises multiple individual units linkedtogether in a polymeric array. Each unit comprises a bridging componentand a complexing agent. In an embodiment, the target molecule complex isformed by combining the selected metal compound, e. g. chromium chloride(III) hexahydrate, with an aqueous buffered solution of the complexingagent. In an embodiment, an aqueous buffered solution of the complexingagent is prepared by dissolving a complexing agent, e.g.,N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, in anaqueous buffered solution, e.g., 10 mM sodium acetate buffer at a finalpH of 3.2-3.3. A metal compound is added in excess in an amountsufficient to complex with an isolatable portion of the complexingagent, and the reaction is conducted at a temperature of approximately20° C. to 33° C. for approximately 24 to 96 hours, or until theresultant complex precipitates out of the aqueous buffered solution. Theprecipitated complex is then isolated for future use.

The precipitated complex is then mixed with the selected lipids or thelipids of the supra-molecular lipid construct and dissolved in anorganic solvent. In an embodiment, the organic solvent ischloroform:methanol (2:1 v/v). The lipids are in a concentrationsufficient to dissolve and incorporate either all or a portion of themetal complex therein. The mixture of the complex and the selectedlipids that form the supra-molecular lipid construct are maintained at atemperature of approximately 60° C. when a high transition temperaturelipid, such as 1,2-distearoyl-sn-glycero-3-phosphocholine, is employed.Lower temperatures may be used depending upon the transition temperatureof the lipids selected for incorporation into the supra-molecular lipidconstruct. A time period from 30 minutes to 2 hours under vacuum isgenerally required to dry the lipids and remove any residual organicsolvent from the lipid matrix in order to form the target moleculecomplex intermediate.

Lipids can be produced and loaded by the methods disclosed herein, andthose methods described in U.S. Pat. Nos. 4,946,787; 4,603,044; and5,104,661, and the references cited therein. Typically, the aqueoussupra-molecular lipid construct formulations of this invention willcomprise 0.1% to 10% active agent by weight (i.e. 1-100 mg drug per ml),and 0.1% to 4% lipid by weight in an aqueous solution, optionallycontaining salts and buffers, in a quantity to make 100% by volume.Preferred are formulations which comprise 0.01% to 5% active agent. Mostpreferred is a formulation comprising 0.01% to 5% active agent by weightand up to 2% by weight of a lipid component in an amount of aqueoussolution sufficient (q. s.) to make 100% by volume.

In an embodiment, glargine insulin was loaded into the target moleculecomplex after the pH of a suspension of the target molecule complex andWater for Injection, USP was adjusted from approximately pH 4.89±0.2 to5.27±0.5. The pH of a solution of glargine insulin was adjusted from pH3.88±0.2 to approximately pH 4.78±0.5, then the water insoluble targetmolecular complex was added. The resulting composition was a mixture offree glargine insulin and glargine insulin associated with a waterinsoluble target molecule complex. A portion of glargine insulin becameassociated with the supra-molecular lipid construct matrix or entrappedin the core volume of the supra-molecular lipid construct. Thispharmaceutical composition is also referred to as HDV-glargine. In anembodiment, an aliquot of the target molecule complex is introduced intoa vial of Glargine Insulin containing 100 International units ofinsulin/ml to provide a hepatocyte specific delivery system containingboth free glargine insulin and glargine insulin associated with thetarget molecule complex.

A pharmaceutical composition that combines free glargine insulin andglargine insulin associated with a water insoluble target moleculecomplex was prepared by the following procedure. The pH of a sample ofSterile Water for Injection, USP, was adjusted to pH 3.95±0.2. Analiquot of HDV suspension was taken and its pH was adjusted in a seriesof steps until the final pH was 5.2±0.5. An aliquot of the Sterile Waterfor Injection, USP, at pH 3.95±0.2 was mixed with the suspension of thetarget molecule complex. The pH of the resulting suspension was4.89±0.2. The pH of this suspension was then adjusted to pH 5.27±0.5.The pH of an aliquot of glargine insulin was adjusted from pH 3.88±0.2to pH 4.78±0.5. This solution was then added to the suspension of thetarget molecule complex at pH 5.20±0.5. The resulting pharmaceuticalcomposition is a mixture of free glargine insulin and glargine insulinassociated with a water insoluble target molecule complex. Thispharmaceutical composition is also referred to as HDV-glargine.

Description of the Invention—Method of Use

Patients with Type I or Type II diabetes are administered an effectiveamount of a hepatocyte targeted composition comprising a mixture of freeglargine insulin and glargine insulin associated with a water insolubletarget molecule complex. In an embodiment, glargine insulin can becombined with other forms of insulin, such as the rapid acting lisproinsulin and insulin aspart, short acting Regular insulin, intermediateacting lente insulin or recombinant human insulin isophane, and longacting ultralente insulin, or premixed combinations of insulins. In anembodiment, the composition can be administered by a subcutaneous ororal route.

After a composition is administered to a patient by subcutaneousinjection, the in situ physiological environment in the injection area,the morphology and chemical structures of free glargine insulin and theglargine insulin associated with the water insoluble target moleculecomplex begin to change. As the pH of the environment around the freeglargine insulin and the glargine insulin associated with the waterinsoluble target molecule complex increases after being diluted withphysiological media, the pH reaches the isoelectric point of glargineinsulin, where flocculation, aggregation and precipitation reactionsoccur for both free glargine insulin and glargine insulin associatedwith the target molecule complex. The rates at which these processesoccur differ between free glargine insulin and glargine insulinassociated with the target molecule complex. The free glargine insulinis directly exposed to changes in pH and dilution. Exposure of glargineinsulin associated with the target molecule complex to small changes inpH and dilution at physiological pH is delayed due to the time requiredfor diffusion of physiological fluids or media through the lipid bilayerin the water insoluble target molecule complex. The delay in the releaseof insulin from the supra-molecular lipid construct as well as the delayof the release of supra-molecular lipid construct with associatedinsulin within the precipitated free glargine matrix is an essentialfeature of this invention since it affects and augments the biologicaland pharmacological response in vivo.

Oral administration of a pharmaceutical composition that combines freeglargine insulin and glargine insulin associated with a target moleculecomplex is followed by intestinal absorption of glargine insulinassociated with the target molecule complex into the circulatory systemof the body where it is also exposed to the physiological pH of theblood. All or a portion of the supra-molecular lipid construct isdelivered to the liver.

As the physiological dilution is increased in situ in the subcutaneousspace or upon entering into the circulatory system, the free glargineinsulin and glargine insulin associated with the target molecule complexencounter a normal physiological pH environment of pH 7.4. As a result,free glargine insulin changes from a soluble form at injection, to ainsoluble form at a pH near its isoelectric point of pH 5.8-6.2, andthen to a soluble form at physiological pH. In the soluble form,glargine insulin migrates through the body to sites where it is capableof eliciting a pharmacological response. Glargine insulin associatedwith the water insoluble target molecule complex becomes solubilized andreleased from the complex at a different rate that is slower than thatof free glargine insulin. This is because glargine insulin associatedwith the water insoluble target molecule complex has to traverse thecore volume and lipid domains of the water insoluble target moleculecomplex before it contacts the bulk phase media.

The supra-molecular lipid construct structure of this invention providesa useful agent for pharmaceutical application for administering glargineinsulin to a host. Accordingly, the structures of this invention areuseful as pharmaceutical compositions in combination withpharmaceutically acceptable carriers. Administration of the structuresdescribed herein can be via any of the accepted modes of administrationfor glargine insulin that are desired to be administered. These methodsinclude oral, parenteral, nasal and other systemic or aerosol forms.

The amount of glargine insulin administered will be dependent on thesubject being treated, the type and severity of the affliction, themanner of administration and the judgment of the prescribing physician.Although effective dosage ranges for specific biologically activesubstances of interest are dependent upon a variety of factors, and aregenerally known to one of ordinary skill in the art, some dosageguidelines can be generally defined. For most forms of administration,the lipid component will be suspended in an aqueous solution andgenerally not exceed 4.0% (w/v) of the total formulation. The drugcomponent of the formulation will most likely be less than 20% (w/v) ofthe formulation and generally greater than 0.01% (w/v).

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 5% with the balance made up from non-toxic carriers may beprepared.

The exact composition of these formulations may vary widely depending onthe particular properties of the drug in question. However, they willgenerally comprise from 0.01% to 5%, and preferably from 0.05% to 1%active ingredient for highly potent drugs, and from 2%-4% for moderatelyactive drugs.

The percentage of active compound contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the activity of the compound and the needs of the subject. However,percentages of active ingredient of 0.01% to 5% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. Preferably thecomposition will comprise 0.2%-2.0% of the active agent in solution.

The formulations of pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the hepatocyte targeted composition into association with acarrier or one or more other ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desired single- ormulti-dose unit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions that aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, parenteral, pulmonary, buccal, or another route ofadministration.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is a discrete amount of apharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage. However, delivery ofthe active agent as set forth in this invention may be as low as 1/10,1/100 or 1/1,000 or smaller than the dose normally administered becauseof the targeted nature of the insulin therapeutic agent.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration may be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active ingredient. Other formulationssuitable for oral administration include, but are not limited to, apowdered or granular formulation, an aqueous or oily suspension, anaqueous or oily solution, or an emulsion.

As used herein, an “oily” liquid is one that comprises acarbon-containing liquid molecule and exhibits a less polar characterthan water.

A tablet comprising the pharmaceutical composition may, for example, bemade by compressing or molding a pharmaceutical composition of theinvention, optionally with one or more additional ingredients.Compressed tablets may be prepared by compressing, in a suitable device,the pharmaceutical composition in a free-flowing form such as a powderor granular preparation, optionally mixed with one or more of a binder,a lubricant, an excipient, a surface active agent, and a dispersingagent. Molded tablets may be made by molding, in a suitable device, amixture of the pharmaceutical composition, a pharmaceutically acceptablecarrier, and at least sufficient liquid to moisten the mixture.Pharmaceutically acceptable excipients used in the manufacture oftablets include, but are not limited to, inert diluents, granulating anddisintegrating agents, binding agents, and lubricating agents. Knowndispersing agents include, but are not limited to, potato starch andsodium starch glycollate. Known surface active agents include, but arenot limited to, sodium lauryl sulphate. Known diluents include, but arenot limited to, calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of thepharmaceutical composition. By way of example, a material such asglyceryl monostearate or glyceryl distearate may be used to coattablets. Further by way of example, tablets may be coated using methodsdescribed in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising a pharmaceutical composition of the currentinvention may be made using a physiologically degradable composition,such as gelatin. Such hard capsules may further comprise additionalingredients including, for example, an inert solid diluent such ascalcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising a pharmaceutical composition of theinvention may further comprise a physiologically degradable composition,such as gelatin. Such soft capsules may further comprise apharmaceutical composition of the invention mixed with water or an oilmedium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionthat are suitable for oral administration may be prepared, packaged, andsold either in liquid form or in the form of a dry product intended forreconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achievesuspension of the pharmaceutical composition in an aqueous or oilyvehicle. Aqueous vehicles include, for example, water and isotonicsaline. Oily vehicles include, for example, almond oil, oily esters,ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconutoil, fractionated vegetable oils, and mineral oils such as liquidparaffin. Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, and hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of a pharmaceutical composition of the invention inaqueous or oily solvents may be prepared in substantially the samemanner as liquid suspensions, the primary difference being that thepharmaceutical composition is dissolved, rather than suspended in thesolvent. Liquid solutions of a pharmaceutical composition of theinvention may comprise each of the components described with regard toliquid suspensions, it being understood that suspending agents will notnecessarily aid dissolution of the pharmaceutical composition in thesolvent. Aqueous solvents include, for example, water and isotonicsaline. Oily solvents include, for example, almond oil, oily esters,ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconutoil, fractionated vegetable oils, and mineral oils such as liquidparaffin.

Powdered and granular formulations of a pharmaceutical composition ofthe invention may be prepared using known methods. Such formulations maybe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations may further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase may be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions may further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of apharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the composition combined with a pharmaceuticallyacceptable carrier, such as sterile water or sterile isotonic saline.Such formulations may be prepared, packaged, or sold in a form suitablefor bolus administration or for continuous administration. Injectableformulations may be prepared, packaged, or sold in unit dosage form,such as in ampules or in multi-dose containers containing apreservative. Formulations for parenteral administration include, butare not limited to, suspensions, solutions, emulsions in oily or aqueousvehicles, pastes, and implantable sustained-release or biodegradableformulations. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, suspending,stabilizing, or dispersing agents. In one embodiment of a formulationfor parenteral administration, the pharmaceutical composition isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

Pharmaceutical compositions may be prepared, packaged, or sold in theform of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the pharmaceutical composition,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally administrableformulations include those that comprise the pharmaceutical compositionin microcrystalline form or as a component of a biodegradable polymersystem. Compositions for sustained release or implantation may comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the pharmaceutical composition and which have a diameter in therange from about 0.5 to about 7 microns, and preferably from about 1 toabout 6 microns. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the pharmaceutical compositiondissolved or suspended in a low-boiling propellant in a sealedcontainer. Preferably, such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 micronsand at least 95% of the particles by number have a diameter less than 7microns. More preferably, at least 95% of the particles by weight have adiameter greater than 1 nanometer and at least 90% of the particles bynumber have a diameter less than 6 microns. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the pharmaceutical composition).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the composition in the form of droplets of asolution or suspension. Such formulations may be prepared, packaged, orsold as aqueous or dilute alcoholic solutions or suspensions, optionallysterile, comprising the pharmaceutical composition, and may convenientlybe administered using any nebulization or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent, or apreservative such as methylhydroxybenzoate. The droplets provided bythis route of administration preferably have an average diameter in therange from about 0.1 to about 200 microns.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the pharmaceutical composition of the invention havingan average particle from about 0.2 to 500 microns. Such a formulation isadministered in the manner in which snuff is taken i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 75% (w/w) ofthe pharmaceutical composition, and may further comprise one or more ofthe additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)pharmaceutical composition, the balance comprising an orally dissolvableor degradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising a pharmaceutical compositionof the invention. Such powdered, aerosolized, or aerosolizedformulations, when dispersed, preferably have an average particle ordroplet size in the range from about 0.1 to about 200 microns, and mayfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1%-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a supra-molecular lipidconstruct preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in pharmaceutical compositions of the invention are known inthe art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, companion animals and othermammals.

Typically dosages of the pharmaceutical composition of the inventionwhich may be administered to an animal, preferably a human, range inamount from 1 microgram to about 1 mg per kilogram of body weight of theanimal. The precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of animal andtype of disease state being treated, the age of the animal and the routeof administration. Preferably, the dosage of the active ingredients inthe composition will vary from about 1 mg to about 10 mg per kilogram ofbody weight of the animal. More preferably, the dosage will vary fromabout 10 mg to about 1 g per kilogram of body weight of the animal.

The composition of the invention may be administered to an animal asfrequently as several times daily, or it may be administered lessfrequently, such as once a day, once a week, once every two weeks, oncea month, or even lees frequently, such as once every several months oreven once a year or less. The frequency of the dose will be readilyapparent to the skilled physician and will depend upon any number offactors, such as, but not limited to, the type and severity of thedisease being treated, the type and age of the animal, etc.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes administeringthe composition to a tissue of a mammal. In another embodiment, this kitcomprises a (preferably sterile) solvent suitable for dissolving orsuspending the composition of the invention prior to administering thecompound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the protein of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the components of the invention or be shipped togetherwith a container which contains the components of the invention.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the instructional material and thecompound be used cooperatively by the recipient.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Experimental Example 1 Pharmaceutical Composition

The materials and methods used in the experiments presented in thisExperimental Example are now described.

A hepatocyte targeted composition comprises a mixture of free glargineinsulin and glargine insulin associated with a water insoluble targetmolecule complex. The complex comprises multiple linked individual unitsand a supra-molecular lipid construct matrix, comprising a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, dicetylphosphate. The bridging agent polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid] is presentwithin the complex.

Experimental Example 2 Preparation of HDV-Glargine Insulin

An intermediate mixture of the components of a target molecule complexwas produced by the following procedure. A mixture of the components[total mass of 2.830 g] of a target molecule complex was prepared byadding aliquots of the lipids 1,2-distearoyl-sn-glycero-3-phosphocholine(2.015 g), crystalline cholesterol (0.266 g), and dicetyl phosphate(0.515 g) to the bridging agent, polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid] (0.034 g).A solution of chloroform (50 ml) and methanol (25 ml) had beendehydrated over molecular sieves. The mixture of the components of thetarget molecule complex was added to the chloroform/methanol solution,which was then placed in a water bath at 60° C.±2° C. to form asolution. The chloroform/methanol solution was removed under vacuum on arotary evaporator using an aspirator, followed by a vacuum pump, and thesolid intermediate mixture formed.

A target molecule complex was produced by the following process. The pHof 530 ml of Sterile Water for Injection, USP (SWI) was adjusted tobetween pH 6.5-7.5 by the addition of a 105 μl of 0.1 N NaOH solution.Sufficient water was added to make 200 g of product. The pH adjusted SWIwas added to the intermediate mixture (2.830 g) and the intermediatemixture was hydrated by placing the mixture in a water bath at 80° C.±2°C. while rotating the mixture for approximately 30 minutes±15 minutes,or until the mixture was a uniform appearing suspension. During theprevious process, the pH of the suspension decreased. The pH of thesuspension was then adjusted to pH 5.44±0.5 pH units by the addition ofapproximately 1.0 ml 0.1 N NaOH.

The suspension of the hydrated target complex was transferred to a modelM-110 EHI microfluidizer that was preheated to 70° C.±10° C. with 28 mMsodium phosphate buffer at pH 7.0. The suspension was microfluidized at9,000 psig using one pass of the suspension of the hydrated targetmolecule complex through the fluidizer. After passing through themicrofluidizer, an unfiltered sample (2.0-5.0 ml) of the fluidizedsuspension was collected for particle size analysis using unimodaldistribution data from a Coulter N-4 plus particle size analyzer. Priorto all particle size determinations, the sample was diluted with 0.2micron filtered SWI that has been pH adjusted to between 6.5-7.5. Theparticle size was required to range from 0.020-0.40 microns. If theparticle size was not within this range, the suspension was passedthrough the microfluidizer again, and the particle size was analyzedagain until the particle size requirements was reached. Themicrofluidized target molecule complex was collected in a sterilecontainer.

The suspension of the microfluidized target molecule complex wasmaintained at 60° C.±2° C. while filtered twice through a sterile 0.8micron+0.2 micron gang filter attached to a 5.0 ml syringe. An aliquotof the filtered suspension was analyzed to determine the particle sizerange of particles in the suspension. The particle size of the final 0.2micron filtered sample was in the range from 0.0200-0.2000 microns, asdetermined from the unimodal distribution printout from the particlesize analyzer. The pH of the filtered suspension of the target moleculecomplex was 3.74±0.2 pH units before pH adjustment. Samples were storedin a refrigerator between 2°-8° C. until further use.

The pharmaceutical composition comprising a mixture of free glargineinsulin and glargine insulin associated with a water insoluble targetmolecule complex, also referred to as HDV-glargine insulin, was producedwas produced by the following process. The pH of a 5.0 ml aliquot of thetwice filtered suspension of the target molecule complex was adjustedfrom an initial pH of pH 3.74±0.2 to pH 5.2±pH 0.5 by the sequentialaddition of sterile 0.1 NaOH according to the following procedure:

-   -   pH 3.74+10 μl 0.1 N NaOH→pH 3.96    -   pH 3.96+20 μl 0.1 N NaOH→pH 4.52    -   pH 4.52+10 μl 0.1 N NaOH→pH 4.69    -   pH 4.69+10 μl 0.1 N NaOH→pH 5.01    -   pH 5.01+10 μl 0.1 N NaOH→pH 5.20

A 1.6 ml aliquot of the target molecule complex suspension at pH5.20±0.5 was combined with 18.4 ml of SWI, which had been adjusted to pH3.95±0.2. The pH of the resulting suspension was adjusted from pH 4.89to pH 5.27±0.5 by the addition of 10 μl±1.0 μl of 0.1 N NaOH.

The pH of 5.0 ml aliquot of Lantus® Glargine—U-100 Insulin was increasedfrom pH 3.88±0.2 to pH 4.78±0.5 by the addition of 60 μl±2 μl of sterile0.1 N NaOH with mixing. A 2.5 ml±0.1 ml aliquot of the target moleculecomplex suspension at pH 5.27±0.5 was added to 5.0 ml±0.1 ml of thesolution of Glargine insulin at pH 4.78±0.5 to produce thepharmaceutical composition containing a mixture of free glargine insulinand glargine insulin associated with the water insoluble target moleculecomplex. The product contained 66.1 IU of glargine insulin/mlsuspension. In an embodiment, the mixture of free glargine insulin andglargine insulin associated with the complex can be produced in a vialof glargine insulin in situ in order to manufacture individual dosageforms.

Example 3 Method of Use of HDV-Glargine Insulin for the Control of BloodGlucose in Tvpe I Diabetes Mellitus Patients

HDV-glargine insulin was administered to patients to determine theability of HDV-glargine insulin to control post prandial blood glucoselevels. Seven Type I diabetes mellitus patients were selected. Thepatients were carefully screened and selected according to criterialisted in the study protocol. The patients were treated with basalglargine insulin and a short-acting insulin at meal times prior toentering the HDV-glargine insulin treatment period. Patients weremonitored (via diary cards and site contact) for four days prior toadministering HDV-glargine insulin to assure that they were inacceptable control of their blood glucose levels. Morning fastingglucose levels were established to be in the range of 100-150 mg/dl.

During the study, the dose of HDV-glargine insulin for each patient was1.2X their usual daily dose of basal glargine insulin to compensate forthe amount of short-acting insulin that they would not receive on thetest days. Blood samples were taken according to a set schedule over 13hours. HDV was added to glargine insulin using the method previouslydescribed to produce a suspension with a final concentration of 66.1 IUglargine/ml and 0.37 mg HDV/ml. The patients were injected withHDV-glargine insulin one hour prior to the morning breakfast. At each ofthe three daily meals, breakfast, lunch and dinner, a 60 gramcarbohydrate meal was prescribed by a dietitian.

The results of the experiments presented in this Experimental Exampleare now described. HDV-glargine insulin was well tolerated by thepatients and no adverse reactions were observed at the injection sites.Hypoglycemic reactions were not observed in patients receiving thistreatment. The blood glucose values of patients treated withHDV-glargine insulin are graphically presented in FIG. 4. FIG. 4 showsthat blood glucose concentrations increased, as anticipated, followingmeals and glucose concentrations decreased over time until the next mealwas eaten. This pattern was observed for all four patients. FIG. 5 showsthe effect of a single dose of HDV-glargine insulin on average bloodglucose concentrations in patients consuming three meals during the day.As with the individual patients, blood glucose concentrations increasedfollowing meals and glucose concentrations decreased over time until thenext meal was eaten. Average blood glucose concentrations were above thebaseline value at all time points. The curve suggests that the efficacyof HDV-glargine insulin improved throughout the day because there wasless variation between the high and low concentrations after the lunchand dinner meals than the breakfast meal. The effect of HDV-glargineinsulin on blood glucose concentrations over time relative to bloodglucose concentrations during fasting are shown in FIG. 6. Blood glucoseconcentrations increased following meals then decreased over timetowards the glucose concentration during fasting until the next meal waseaten. Blood glucose concentrations were above fasting concentrationsthroughout the study. Treatment of patients with HDV-glargine insulinresulted in some degree of post-prandial blood glucose level control,indicating that HDV was able to carry sufficient quantities ofglargine-insulin to the liver at mealtimes to provide this control.Blood glucose levels were typical of Type I patients that usuallyreceive basal insulin therapy plus short-acting insulins at meal times.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1. A hepatocyte-targeting composition comprising: a. free glargineinsulin; and b. glargine insulin associated with a water-insolubletarget molecule complex; wherein said target molecule complex iscomprised of a combination of: a. multiple linked individual units, saidindividual units comprising: i. at least one bridging component selectedfrom the group consisting of a transition element, an inner transitionelement, and a neighbor element of said transition element; and ii. acomplexing component; and b. a supra-molecular lipid construct matrixcomprising at least one lipid component; provided that when saidtransition element is chromium, a chromium target molecule complex iscreated; further wherein said target molecule complex comprises anegative charge.
 2. The hepatocyte targeted composition of claim 1,further comprising at least one free non-glargine insulin and at leastone non-glargine insulin associated with a water-insoluble targetmolecule complex.
 3. The hepatocyte targeted composition of claim 2,wherein said non-glargine insulin is selected from the group consistingof lispro insulin, aspart insulin, regular insulin, lente insulin,ultralente insulin, recombinant human regular insulin, recombinant humaninsulin isophane or premixed combinations of any of the aforementionedinsulins, and a combination of two or more of the aforementionedinsulins.
 4. The hepatocyte targeted composition of claim 2, whereinsaid non-glargine insulin comprises insulin-like moieties, includingfragments of insulin molecules, that have the biological activity ofinsulins.
 5. The hepatocyte-targeting composition of claim 1, whereinsaid lipid component comprises at least one lipid selected from thegroup consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine, cholesterol, cholesterololeate, dicetylphosphate, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dimyristoyl-sn-glycero-3-phosphate.
 6. The hepatocyte-targetingcomposition of claim 1, wherein said lipid component comprises at leastone lipid selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dicetylphosphate.
 7. The hepatocyte-targeting composition of claim 1, whereinsaid lipid component comprises a mixture of1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and dicetylphosphate.
 8. The hepatocyte-targeting composition of claim 1, whereinsaid bridging component is chromium.
 9. The hepatocyte-targetingcomposition of claim 1, wherein said complexing component comprises atleast one member selected from the group consisting of:N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid; N-(4-tertiarybutylphenylcarbamoylmethyl) iminodiacetic acid;N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid; aminopyrroliminodiacetic acid; N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl)iminodiacetic acid; benzimidazole methyl iminodiacetic acid;N-(3-cyano-4,5-dimethyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid;N-(3-cyano-4-methyl-5-benzyl-2-pyrrylcarbamoylmethyl) iminodiaceticacid; and N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiaceticacid.
 10. The hepatocyte-targeting composition of claim 1, wherein saidcomplexing component comprisespoly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid].11. A method of manufacturing a hepatocyte targeted composition of claim1 comprising: creating a target molecule complex, wherein said complexcomprises multiple linked individual units and a supra-molecular lipidconstruct matrix; forming a suspension of the target molecule complex inwater; adjusting the pH of said water suspension to approximately pH5.3; adjusting the pH of said glargine insulin to approximately 4.8; andcombining said glargine insulin, said non-glargine insulin and saidtarget molecule complex.
 12. A method of treating a patient for Type Ior Type II diabetes comprising administering to the patient an effectiveamount of a hepatocyte targeted composition of claim
 1. 13. The methodof treating a patient according to claim 12, wherein the route ofadministration is selected from the group consisting of oral,parenteral, subcutaneous, pulmonary and buccal.
 14. The method oftreating a patient according to claim 12, wherein the route ofadministration is oral or subcutaneous.
 15. A method of treating apatient for Type I or Type II diabetes comprising administering to thepatient an effective amount of a hepatocyte targeted composition ofclaim 2, wherein said non-glargine insulin is selected from the groupconsisting of lispro insulin, aspart insulin, regular insulin, lenteinsulin, ultralente insulin, recombinant human regular insulin,recombinant human insulin isophane or premixed combinations of any ofthe aforementioned insulins, and a combination of two or more of theaforementioned insulins.
 16. The method of treating a patient accordingto claim 12, wherein said non-glargine insulin comprises insulin-likemoieties, including fragments of insulin molecules, that have biologicalactivity of insulins.
 17. A method of increasing the bioavailability ofglargine insulin in a patient, said method comprising: administeringglargine insulin in a hepatocyte-targeting composition, said compositioncomprising free glargine insulin and glargine insulin associated with awater insoluble target molecule complex, wherein said complex comprisesmultiple linked individual units and a supra-molecular lipid constructmatrix containing a negative charge, said multiple linked individualunits comprising: (a) a bridging component selected from the groupconsisting of a transition element, an inner transition element, aneighbor element of said transition element and a mixture of any of theforegoing elements, (b) a complexing component, provided that when saidtransition element is chromium, a chromium target molecule complex iscreated, wherein said multiple linked individual units are combined withsaid supra-molecular lipid construct matrix, wherein said insulins areassociated with said target molecule complex that contains a negativecharge; thereby the association between glargine insulin and said waterinsoluble target molecule complex is altered within said patient to formnew structures associated with said glargine insulin, wherein said newstructures are present in soluble and insoluble forms and are deliveredto sites of insulin activity.
 18. The method of increasing thebioavailability of glargine insulin in a patient according to claim 17,wherein glargine insulin is delivered to fat, liver, and muscle.
 19. Themethod of increasing the bioavailability of glargine insulin in apatient according to claim 17, wherein delivery of glargine insulin tosites of insulin activity occurs over a plurality of meals.
 20. Themethod of increasing the bioavailability of glargine insulin in apatient according to claim 17, wherein delivery of glargine insulin tosites of insulin activity comprises a hepatic component delivered atmeal-time.
 21. A kit for treating Type I or Type II diabetes in amammal, said kit comprising glargine insulin and a water insolubletarget molecule complex, wherein said complex comprises multiple linkedindividual units and a supra-molecular lipid construct matrix containinga negative charge, said multiple linked individual units comprising: abridging component selected from the group consisting of a transitionelement, an inner transition element, a neighbor element of saidtransition element and a mixture of any of the foregoing elements, and acomplexing component, provided that when said transition element ischromium, a chromium target molecule complex is created, wherein saidmultiple linked individual units are combined with said supra-molecularlipid construct matrix, wherein the glargine insulin is associated witha target molecule complex, wherein said complex comprises a negativecharge, said kit further comprising a physiological buffered solution,an applicator, and an instructional material for the use thereof. 22.The kit of claim 21 further comprising at least one non-glargineinsulin.